JP5992135B2 - Process for producing lactic acid by continuous fermentation - Google Patents

Description

  The present invention stabilizes yeast culture and fermentation by making yeast capable of producing lactic acid into a higher-order polyploid, and enables production of lactic acid efficiently for a long period of time. Production of lactic acid by continuous fermentation Regarding the method.

  Fermentation methods can be broadly classified into batch fermentation methods (Batch fermentation methods), fed-batch fermentation methods (Fed-Batch fermentation methods), and continuous fermentation methods. The batch fermentation method and fed-batch fermentation method are simple in terms of equipment and have an advantage of less damage due to contamination by various bacteria because the culture is completed in a short time. However, when the concentration of the product in the culture solution increases with the passage of time, there is a problem that productivity and yield decrease due to the influence of osmotic pressure or product inhibition. Therefore, it is difficult to stably maintain a high yield and high productivity over a long period of time. The continuous fermentation method has the advantage of maintaining a high yield and high productivity over a long period of time by avoiding accumulation of the target substance at a high concentration in the fermenter. It is very difficult to continue the culture by the method for a long period of time, and research has been repeated.

  Proposals for continuous fermentation methods include filtration of microorganisms and cultured cells through a separation membrane, collecting the product from the filtrate, and simultaneously holding or refluxing the filtered microorganisms and cultured cells in the culture solution. There are ways to keep the cell concentration high.

  For example, techniques for continuous fermentation in a continuous fermentation apparatus using a ceramic membrane are disclosed (Patent Documents 1 to 3). However, the disclosed technology has a problem in reduction of filtration flow rate and filtration efficiency due to clogging of the ceramic film, and back cleaning or the like is performed to prevent clogging.

  Moreover, the manufacturing method (patent document 5) by continuous fermentation of succinic acid is disclosed. In this technique, a high filtration pressure (about 200 kPa) is employed in membrane separation. The high filtration pressure is not only disadvantageous in terms of cost, but also the microorganisms and cells are physically damaged by the pressure during the filtration process. Not appropriate. Furthermore, in Patent Document 5, techniques such as a separation membrane and filtration pressure are disclosed as proposals for continuing continuous fermentation for a long period of time. However, continuous fermentation time is about 300 hours, and continuous culture is performed for a longer period of time. The proposal of the method of continuing is desired.

  On the other hand, in the examination of microorganisms used for the production of organic acids, studies using yeast having strong resistance to acids have been actively conducted (Non-patent Documents 1 and 2). In yeast, in addition to haploid yeast in which only one set of chromosomes exists, there is a higher-order polyploid having a plurality of sets of chromosomes. High-order polyploid yeast is mainly used as baker's yeast or brewing yeast (Patent Documents 6 to 8). These are used for the production of food and beverages, and are used for the purpose of improving the flavor and manufacturing process, and there is no description used for continuous culture.

  Moreover, the manufacturing method of lactic acid using a higher order polyploid is disclosed (patent documents 9 to 12). These use higher-order polyploids to increase the number of lactic acid synthesis genes, but they are all cultured by the Fed-Batch fermentation method, and the culture time is as short as 100 hours or less. There is no description of continuous fermentation using a membrane.

  In addition, lactic acid production using non-nutrient-requiring yeast that does not have auxotrophy has been reported (Patent Document 13), but the culture time is also less than 100 hours, and the fermentation method is also Fed-Batch. Only the fermentation method is disclosed.

Thus, the continuous fermentation method for increasing productivity and the microorganisms used for fermentation have been studied separately. When microorganisms are cultured by a continuous fermentation method, which is advantageous as a fermentation method, the filtration pressure increases with the culture and it becomes impossible to continue for a long time, or the productivity of lactic acid decreases as the culture is prolonged. Thus, it was difficult to fully demonstrate the advantages of both. Therefore, it has been desired to develop a continuous fermentation technique that can solve these problems and can stably produce lactic acid for a long period of time.
JP-A-5-95778 Japanese Patent Laid-Open No. 62-138184 JP-A-10-174594 JP 2005-333886 A JP 2007-252367 A JP 2000-139326 A JP 2002-027974 A JP 2002-253212 A JP 2006006271 A JP 2006-020602 A JP 2007-089466 A JP 2001-204464 A US200501127737 Biotechnology progress, 11,294-298 (1995) Journal of fermentation and bioengineering, 86, 284-289 (1988)

  The objective of this invention is providing the manufacturing method of lactic acid by the continuous fermentation method which can maintain the high productivity of lactic acid stably over a long time.

As a result of diligent research, the present inventors have determined that in order to maintain high productivity of lactic acid while lactic acid-producing yeast stably repeats growth for a long period of time in continuous fermentation, yeast having lactic acid-producing ability is a higher order multiple. As a result, it was found that high productivity of lactic acid can be maintained for a long period of time, and the present invention has been completed. That is, the present invention has the following configuration.

(1) A high-order polyploidy yeast -free nutrient solution having the ability to produce lactic acid introduced with a lactate dehydrogenase gene is a porous membrane having an average pore size of 0.01 μm or more and less than 1 μm. A method for producing lactic acid by continuous fermentation, characterized by filtering, recovering a product from the filtrate, holding or refluxing the unfiltrated liquid in the culture solution, and adding a fermentation raw material to the culture solution.

  (2) The method for producing lactic acid according to (1), wherein the transmembrane differential pressure of the porous membrane is filtered within a range of 0.1 kPa to less than 20 kPa.

  (3) The method for producing lactic acid according to (1) or (2), wherein the higher order polyploid yeast is diploid.

( 4 ) The method for producing lactic acid according to any one of (1) to ( 3 ), wherein the yield of lactic acid to sugar in continuous fermentation is 70% or more.

( 5 ) The method for producing lactic acid according to any one of (1) to ( 4 ), wherein the concentration of lactic acid in the culture solution of continuous fermentation is 40 g / L or more.

( 6 ) The method for producing lactic acid according to any one of (1) to ( 5 ), wherein the lactic acid production rate of continuous fermentation is 7.5 g / L / h or more.

( 7 ) The method for producing lactic acid according to any one of ( 4 ) to ( 6 ), wherein continuous fermentation is continued for 400 hours or more.

(8) yeast is characterized in that belongs to Saccharomyces (Saccharomyces) genus (1) to method for producing lactic acid according to any one of (7).

(9) yeast is characterized in that it is a Saccharomyces Serebise (Saccharomyces cerevisiae) (1) ~ method for producing lactic acid according to any one of (8).

  According to the present invention, by using a higher-order polyploid yeast, continuous fermentation that stably maintains high productivity of lactic acid that is a desired fermentation product over a long period of time is possible, and lactic acid can be stably produced at low cost. Production is also possible.

FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus used in the present invention. FIG. 2 is a schematic side view for explaining one embodiment of another membrane separation type continuous fermentation apparatus used in the present invention. FIG. 3 is a schematic perspective view for explaining one embodiment of the separation membrane element used in the present invention. FIG. 4 is a schematic perspective view for explaining an example of another separation membrane element used in the present invention. FIG. 5 is a vector for expressing lactate dehydrogenase gene. FIG. 6 shows changes in lactic acid accumulation concentration in Examples 13 and 14, and Comparative Examples 16 and 17. FIG. 7 shows changes in the yield of lactic acid versus sugar in Examples 13 and 14, and Comparative Examples 16 and 17. FIG. 8 shows changes in the lactic acid production rate in Examples 13 and 14, and Comparative Examples 16 and 17. FIG. 9 shows changes in the lactic acid accumulation concentration in Examples 15 and 16, and Comparative Examples 18 and 19. FIG. 10 shows changes in the yield of lactic acid versus sugar in Examples 15 and 16 and Comparative Examples 18 and 19. FIG. 11 shows changes in the production rate of lactic acid in Examples 15 and 16 and Comparative Examples 18 and 19.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fermentation reaction tank 2 Separation membrane element 3 Water head difference control device 4 Gas supply device 5 Stirrer 6 Level sensor 7 Medium supply pump 8 pH adjustment solution supply pump 9 pH sensor / control device 10 Temperature controller 11 Fermentation liquid circulation pump 12 Membrane separation Tank 13 Support plate 14 Channel material 15 Separation membrane 16 Concave portion 17 Water collecting pipe 18 Separation membrane bundle 19 Upper resin sealing layer 20 Lower resin sealing layer 21 Support frame 22 Water collection pipe

  The present invention is a method for producing lactic acid by continuous fermentation while maintaining high lactic acid productivity for a long period of time by using a higher order polyploid yeast in fermentation for culturing yeast having the ability to produce lactic acid. A culture solution of higher-order polyploid yeast having the ability to produce lactic acid is filtered through a separation membrane, the product is recovered from the filtrate, and the unfiltered solution is retained or refluxed in the culture solution, and In the continuous fermentation in which the fermentation raw material is added to the culture solution, a method for producing lactic acid, which uses a porous membrane having an average pore diameter of 0.01 μm or more and less than 1 μm as a separation membrane.

  First, a porous membrane used as a separation membrane in the present invention will be described. The porous membrane in the present invention preferably has separation performance and water permeability according to the quality of water to be treated and the application. The porous membrane is preferably a porous membrane including a porous resin layer from the viewpoint of blocking performance, water permeability performance and separation performance, for example, stain resistance.

  The porous membrane including the porous resin layer preferably has a porous resin layer that acts as a separation functional layer on the surface of the porous substrate. Here, the material of the porous substrate is made of an organic material and / or an inorganic material, and preferably an organic material, more preferably an organic fiber. Among them, a preferable porous substrate is a woven or non-woven fabric using organic fibers such as cellulose fiber, cellulose triacetate fiber, polyester fiber, polypropylene fiber and polyethylene fiber, and more preferably, the density control is relatively easy. There are non-woven fabrics that are easy to manufacture and inexpensive. The thickness of the porous substrate is preferably 50 μm or more and 3000 μm or less in order to support the porous resin layer and give strength to the separation membrane. In addition, the porous resin layer may be permeated into the porous base material or the porous resin layer may not be permeated into the porous base material.

  As the porous resin layer, an organic polymer film can be preferably used. Examples of the material of the organic polymer film include polyethylene resin, polypropylene resin, polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin, polyacrylonitrile resin, polyolefin resin, A cellulose resin, a cellulose triacetate resin, etc. are mentioned, The mixture of resin which has these resins as a main component may be sufficient. Here, the main component means that the component is contained in an amount of 50% by weight or more, preferably 60% by weight or more. In particular, the organic polymer film is made of a polyvinyl chloride resin, a polyvinylidene fluoride resin, a polysulfone resin, or a polyether sulfone resin that is easy to form into a film and has excellent physical durability and chemical resistance. A resin, a polyacrylonitrile-based resin, a polyolefin-based resin, or a mixture of these resins as a main component is preferable, and a polyvinylidene fluoride-based resin or a mixture of resins having a main component thereof is more preferably used.

Here, as the polyvinylidene fluoride resin, a homopolymer of vinylidene fluoride or a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride is preferably used. The vinylidene fluoride and copolymerizable vinyl monomer, tetrafluoroethylene, etc. hexafluoropropylene Pile emission is exemplified.

  Examples of the polyolefin resin include polyethylene, polypropylene, chlorinated polyethylene, and chlorinated polypropylene, and chlorinated polyethylene is preferably used.

  It is important that the porous membrane used in the present invention has a surface average pore diameter of 0.01 μm or more. When the average pore diameter on the surface of the porous membrane is 0.01 μm or more, clogging due to bacterial cells used for fermentation is unlikely to occur, and the filtration performance is stable for a long period of time. In addition, when the average pore diameter of the porous membrane is 0.01 μm or more, it is possible to achieve both a high exclusion rate at which the higher order polyploid yeast does not leak and a high water permeability, and the water permeability can be maintained for a long time. Holding can be performed with higher accuracy and reproducibility. In addition, when the average pore diameter on the surface of the porous membrane in the present invention is less than 0.01 μm, the water permeability of the porous membrane is lowered, and efficient operation may not be possible even if the membrane is not dirty. When the average pore diameter of the porous membrane is 0.01 μm or more, preferably 0.02 μm or more, more preferably 0.04 μm or more, efficient operation is possible.

The average pore diameter of the porous membrane in the present invention is to prevent leakage of high-order polyploid yeast, that is, the occurrence of a problem that the rejection rate decreases, and also prevent high-order polyploid yeast from directly blocking the pores. Therefore, it is important that the thickness is less than 1 μm, and it is preferably 0.4 μm or less, and more preferably 0.2 μm or less. In addition, substances other than the target lactic acid such as proteins and polysaccharides may be produced by the higher-order polyploid yeast, and some of the higher-order polyploid yeasts in the culture medium are killed. In this case, crushed cells may be generated, and in order to avoid the porous membrane from being blocked by these substances, it is more preferable that the average pore diameter is 0.1 μm or less. Thus, the average pore diameter of the surface of the porous membrane used in the present invention, 0 .4Myuemu or less, and more preferably at most 0.2 [mu] m, and particularly preferably 0.1 [mu] m.

  Here, the average pore diameter of the surface in the present invention is the diameter of all pores that can be observed within the range of 9.2 μm × 10.4 μm in the observation of the scanning electron microscope porous membrane surface at a magnification of 10,000 times. It can be determined by measuring and averaging. The average pore diameter is obtained by taking a photograph of the membrane surface at a magnification of 10,000 using a scanning electron microscope, and randomly selecting 10 or more, preferably 20 or more pores. It can also be obtained by measuring and number average. When the pores are not circular, a circle having an area equal to the area of the pores (equivalent circle) is obtained by an image processing device or the like, and the equivalent circle diameter is obtained by the method of setting the diameter of the pores.

  The average pore diameter of the surface of the porous membrane used in the present invention has a smaller standard deviation of the pore diameter, that is, a uniform permeate can be obtained when the pore diameter is uniform, and the standard deviation σ is It is preferably 0.1 μm or less. Moreover, since the fermentation operation management becomes easy, the smaller the standard deviation of the average pore diameter, the better. The standard deviation σ of the average pore diameter was measured using N as the number of pores that can be observed within the range of 9.2 μm × 10.4 μm in the observation of the scanning electron microscope porous film surface at the magnification of 10,000. It is calculated by the following (Formula 1) where each diameter is Xk and the average pore diameter is X (ave).

In the porous membrane used in the present invention, the permeability of the culture solution is one of the important performances. As an index of the permeability of the porous membrane, the pure water permeability coefficient of the porous membrane before use can be used. In the present invention, the pure water permeability coefficient of the porous membrane is obtained by filtering drinking water with a dialysis membrane (Toray Co., Ltd., Filtizer B2-1.5H) as raw water, at 25 ° C. and a head height of 1 m. When the water permeability is measured and calculated, it is preferably 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more, and the pure water permeability coefficient is 2 × 10 ⁻⁹ m ³ / m ² / s / pa. If it is 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less, a practically sufficient amount of permeated water can be obtained. More preferably, they are 2 * 10 < ^-9 > m < ³ > / m < ² > / s / pa or more and 1 * 10 < ^-7 > m < ³ > / m < ² > / s / pa or less.

  The membrane surface roughness in the porous membrane used in the present invention is an average value of heights in the direction perpendicular to the surface. Membrane surface roughness is one of the factors that make it easier for exfoliated yeast adhering to the surface of the separation membrane to peel off due to the membrane surface cleaning effect by liquid flow using agitation and circulation pump. The surface roughness of the porous membrane is preferably 0.1 μm or less. When the membrane surface roughness is 0.1 μm or less, the higher-order polyploid yeast attached to the membrane is easy to peel off, and in the filtration of the higher-order polyploid yeast, the shear force generated on the membrane surface can be reduced. Is prevented, and clogging of the porous membrane is suppressed, so that stable filtration is possible for a long period of time, and continuous fermentation can be carried out with a lower transmembrane pressure difference, so the membrane is clogged. Even in this case, the cleaning recoverability is better than the case of operating at a high transmembrane pressure. In addition, since stable continuous fermentation becomes possible by suppressing clogging, the surface roughness of a porous membrane is so preferable that it is small.

  Here, the film surface roughness is measured using the following atomic force microscope (AFM) under the following conditions.

Apparatus: Atomic force microscope (Digital Instruments Co., Nanoscope IIIa)
Condition Probe: SiN cantilever (manufactured by Digital Instruments)
Scan mode Contact mode (in-air measurement)
Underwater tapping mode (underwater measurement)
Scanning range: 10μm, 25μm square (measurement in air)
5μm, 10μm square (underwater measurement)
Scanning resolution: 512 × 512
Sample preparation: For measurement, the membrane sample is immersed in ethanol at room temperature for 15 minutes, then immersed in RO water for 24 hours, washed, and then air-dried.

  The film surface roughness (Drough) is calculated from the height in the Z-axis direction of each point by the above-described atomic force microscope (AFM) by the following (Formula 2).

  The shape of the porous membrane used in the present invention may be a flat membrane or a hollow fiber membrane. When the shape of the porous membrane is a flat membrane, the average thickness is selected according to the application, but is preferably 20 μm or more and 5000 μm or less, more preferably 50 μm or more and 2000 μm or less. When the porous membrane is a hollow fiber membrane, the inner diameter of the hollow fiber is preferably 200 μm or more and 5000 μm or less, and the film thickness is preferably 20 μm or more and 2000 μm or less. Further, a woven fabric or a knitted fabric in which organic fibers or inorganic fibers are formed in a cylindrical shape may be included in the hollow fiber.

  The method for producing the porous membrane used in the present invention will be described by way of example.

  First, a method for producing a flat film which is one of the preferred embodiments of the porous film will be described. A film of a stock solution containing a resin and a solvent is formed on the surface of the porous base material, and the porous base material is impregnated with the stock solution. Thereafter, only the coating-side surface of the porous substrate having a coating is brought into contact with a coagulation bath containing a non-solvent to solidify the resin and form a porous resin layer on the surface of the porous substrate.

Stock is made tone by dissolving a resin in a solvent. The temperature of the stock solution is usually preferably selected within the range of 5 to 120 ° C. from the viewpoint of film forming properties. The solvent dissolves the resin and acts on the resin to encourage them to form a porous resin layer. The solvent, N- methylpyrrolidinone (NMP), N, N- dimethylacetamide (DMAc), N, N- dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N- methyl-2-pyrrolidone, Te tetrahydrofuran , Tetramethylurea, trimethyl phosphate, cyclohexanone, isophorone, γ-butyrolactone, methyl isoamyl ketone, dimethyl phthalate, propylene glycol methyl ether, propylene carbonate, diacetone alcohol, glycerol triacetate, acetone and methyl ethyl ketone can be used . Of these, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), and dimethylsulfoxide (DMSO), which have high resin solubility, can be preferably used. These may be used alone or in combination of two or more.

  In addition, components other than solvents such as polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and glycerin may be added to the solvent. Non-solvents can also be added to the solvent. The non-solvent is a liquid that does not dissolve the resin. The non-solvent acts to control the pore size by controlling the rate of solidification of the resin. As the non-solvent, water and alcohols such as methanol and ethanol can be used. Of these, water and methanol are preferable as the non-solvent from the viewpoint of cost. Components other than the solvent and the non-solvent may be a mixture.

  A pore opening agent may be added to the stock solution. The pore-opening agent is extracted when immersed in the coagulation bath, and has a function of making the resin layer porous. By adding a pore opening agent, the average pore size can be controlled. The pore-opening agent is preferably one having high solubility in the coagulation bath. As the pore opening agent, for example, an inorganic salt such as calcium chloride or calcium carbonate can be used. As the pore opening agent, polyoxyalkylenes such as polyethylene glycol and polypropylene glycol, water-soluble polymer compounds such as polyvinyl alcohol, polyvinyl butyral and polyacrylic acid, and glycerin can be used.

  Next, a method for producing a hollow fiber membrane which is one of the preferred embodiments of the porous membrane will be described. The hollow fiber membrane discharges a stock solution composed of a resin and a solvent from the outer tube of the double-tube base, and discharges a hollow portion forming fluid from the inner tube of the double-tube base in a cooling bath. It can be produced by cooling and solidifying.

Stock solution at a concentration resin following 20 wt% to 60 wt% as described in preparation method of the aforementioned flat membrane, it flat-film tone that is dissolved in the solvent mentioned in generation method described above. Moreover, a gas or a liquid can be normally used for the hollow portion forming fluid. In addition, a new porous resin layer can be coated (laminated) on the outer surface of the obtained hollow fiber membrane. Lamination can be performed in order to change the properties of the hollow fiber membrane, for example, hydrophilicity / hydrophobicity, pore diameter and the like into desired properties. A new porous resin layer to be laminated can be produced by bringing a stock solution obtained by dissolving a resin in a solvent into contact with a coagulation bath containing a non-solvent to coagulate the resin. As the material of the resin, for example, the same material as that of the organic polymer film can be preferably used. The method of lamination is not particularly limited, and the hollow fiber membrane may be immersed in the stock solution, or the stock solution may be applied to the surface of the hollow fiber membrane, and after lamination, a part of the deposited stock solution is scraped off. Or the amount of stacking can be adjusted by blowing off with an air knife.

  The porous membrane used in the present invention can be made into a separation membrane element by adhering / sealing the hollow portion of the hollow fiber membrane using a member such as a resin and setting it on a support.

  The porous membrane used in the present invention can be made into a separation membrane element by combining with a support. A separation membrane element in which a support plate is used as a support and the porous membrane used in the present invention is disposed on at least one surface of the support plate is a preferred embodiment of the separation membrane element having a porous membrane used in the present invention. One. In order to increase the amount of water permeation, a preferred embodiment of the separation membrane element is to dispose a porous membrane on both sides of the support plate.

  In the method for producing lactic acid according to the present invention, it is preferable to perform filtration treatment in a range where the pressure difference between the porous membranes is 0.1 kPa or more and less than 20 kPa. In order to filter the fermentation broth, when filtration is performed at a transmembrane pressure of 20 kPa or more, power for applying pressure is required, and the economic effect when producing lactic acid is lowered. Moreover, by applying a higher transmembrane pressure of 20 kPa or more, higher-order polyploid yeast used for continuous fermentation culture may be crushed, and the ability to produce lactic acid is reduced. When the transmembrane pressure is less than 0.1 kPa, it takes time for filtration and the production rate is lowered. If the transmembrane differential pressure, which is the filtration pressure, is in the range of 0.1 or more and less than 20 kPa, the transmembrane differential pressure can be obtained by the water head difference. The ability to produce will not decline. Moreover, since it is not necessary to keep the inside of a fermenter in a pressurized state specially, the aspect which installs a porous membrane inside a fermenter is also attained, and the advantage that a fermenter can be made compact is also mentioned.

  The transmembrane differential pressure here refers to the pressure difference between the treated water side and the permeated water side in the porous membrane. When operation is performed with a constant treatment flow rate per unit area of the porous membrane, if the water to be treated is continuously filtered through the porous membrane for a long period of time, the contaminants present in the water to be treated are removed from the porous membrane. The membrane is contaminated due to being adsorbed by the pores and deposited on the surface. Then, membrane clogging occurs, the transmembrane pressure difference is improved, the treatment flow rate is remarkably lowered, and it is difficult to continue stable operation. Therefore, it is preferable to perform filtration while measuring the transmembrane pressure difference. The method for measuring the transmembrane pressure can be measured by installing pressure gauges on the treated water side and the permeate side in the porous membrane, measuring the pressure, and determining the pressure difference.

  Next, the high-order polyploid yeast having the ability to produce lactic acid used in the present invention will be described. In the method for producing lactic acid according to the present invention, by using a high-order polyploid yeast having an ability to produce lactic acid, high productivity of lactic acid which is a desired fermentation product stably over a long period of time under simple operation conditions. Thus, it becomes possible to continuously produce lactic acid at a low cost.

  The higher order polyploid yeast is a yeast having two or more sets of chromosomes in a cell. Higher polyploid yeasts are larger than haploid yeasts generally used for genetic analysis, so that the porous membrane is less likely to block and is suitable for long-term culture. By using higher-order polyploid yeast, the shear force generated on the membrane surface can be reduced, the destruction of the yeast is suppressed, and clogging of the porous membrane is also suppressed. As well as being possible, the membrane can be reused, and the cost can be reduced. Further, even when the membrane is clogged, the washing recovery property is better than that of haploid yeast. There is no particular restriction on the number of chromosomes of higher order polyploid yeast, but diploid yeast having two sets of chromosomes is preferred.

Examples of the higher polyploid yeast include baker's yeast, sake yeast, wine yeast, beer yeast and the like often used in the fermentation industry. Higher polyploid yeast used may be those isolated from natural environment, or may be one part properties modified by mutation or gene recombination.

The higher polyploid yeast having the ability to produce lactic acid, lactate dehydrogenase gene (hereinafter, sometimes referred to as LDH.) Is introduced. There is no particular limitation on the method for producing a high-order polyploid yeast into which lactate dehydrogenase has been introduced, but by converting a haploid yeast into which a lactate dehydrogenase gene has been introduced onto a chromosome to higher-order polyploidy, Without changing the genotype per chromosome, it is possible to easily increase the number of copies of the lactate dehydrogenase gene, thereby enhancing the ability to produce lactate.

  Furthermore, in the present invention, if a higher-order polyploid yeast having the ability to produce lactic acid is not nutrient-neutral, a medium with fewer nutrients than before, that is, a low-cost medium can be used, and simple operation is possible. Under the conditions, continuous fermentation that stably maintains high productivity of lactic acid over a long period of time is possible, and lactic acid can also be produced stably at low cost, so it is preferably used in the present invention.

  The auxotrophy possessed by yeast means that a mutation has occurred in the nutrient synthesis gene of wild-type yeast for some reason, and as a result, it lacks the ability to synthesize the nutrient. In addition, restoration of auxotrophy means that the auxotrophy mutation is restored to the wild type or very close to the wild type. Since auxotrophy is mainly used as a marker during genetic manipulation or the like, auxotrophic yeast is preferably used during genetic manipulation.

  When yeast has auxotrophy, specific examples of necessary nutrients include methionine, tyrosine, isoleucine, phenylalanine, glutamic acid, threonine, aspartic acid, valine, serine, arginine, uracil, adenine, lysine, tryptophan, leucine, Histidine and the like are known. Examples of the genotype of yeast exhibiting auxotrophy include the following.

Methionine requirement: met1, met2, met3, met4, met5, met6, met7, met8, met10, met13, met14, met20
・ Tyrosine requirement: tyr1
Isoleucine, valine requirement: ilv1, ilv2, ilv3, ilv5
-Phenylalanine requirement: pha2
・ Glutamic acid requirement: GLU3
・ Threonine requirement thr1, thr4
Aspartic acid requirement: asp1, asp5
Serine requirement: ser1, ser2
Arginine requirement: arg1, arg3, arg4, arg5, arg8, arg9, arg80, arg81, arg82, arg84
・ Uracil requirement: ura1, ura2, ura3, ura4, ura6
Adenine requirement: ade1, ade2, ade3, ade4, ade5, ade6, ade8, ade9, ade12, ADE15
Lysine requirement: lys1, lys2, lys4, lys5, lys7, lys9, lys11, lys13, lys14
Tryptophan requirement: trp1, trp2, trp3, trp4, trp5
・ Leucine requirement: leu1, leu2, leu3, leu4, leu5
Histidine requirement: his1, his2, his3, his4, his5, his6, his7, his8.

  An auxotrophic yeast preferably used in the present invention is a yeast that does not have a genotype exhibiting the above auxotrophy or is complemented. In addition, as a method for determining whether or not a yeast is a non-nutrient requirement yeast, it can be used as a criterion for judgment based on whether or not the yeast can grow on an SD medium (Table 1), which is a minimum yeast culture medium.

  To restore the auxotrophy of auxotrophic yeast and produce non-nutrient auxotrophic yeast, it is possible to restore the auxotrophy by introducing a nutritional synthesis gene by genetic recombination techniques, or different auxotrophy. Examples include a method in which yeasts having a yeast are joined to form ascoles and the target auxotrophy is restored, and finally all the auxotrophy is restored to obtain an auxotrophic yeast.

  As LDH introduced in higher polyploid yeast having the ability to produce lactic acid, reduced nicotinamide adenine dinucleotide (NADH) and pyruvate are converted to oxidized nicotinamide adenine dinucleotide (NAD +) and lactic acid. For example, LDH derived from lactic acid bacteria having a high yield of lactic acid against saccharide, LDH derived from mammals, or LDH derived from amphibians can be used. Of these, LDH derived from Homo sapiens and frog is preferably used. Furthermore, it is preferable to use LDH derived from a frog belonging to the family Pipidae among frogs, and LDH derived from Xenopus laevis can be preferably used from the frogs belonging to the family Frogidae.

  The LDH includes genetic polymorphisms and mutated genes caused by mutagenesis. Here, genetic polymorphism means that the base sequence of a gene is partly changed due to natural mutation on the gene, and mutagenesis means that a mutation is artificially introduced into the gene. Mutagenesis is, for example, a method using a site-directed mutagenesis kit (Mutan-K (Takara Bio) or a random mutagenesis kit (BD Diversify PCR Random Mutagenesis (CLONTECH)). There is. In addition, the LDH used in the present invention may have a deletion or insertion in a part of the base sequence as long as it encodes a protein having an activity of converting NADH and pyruvate into NAD + and lactic acid. Absent.

  Higher polyploid yeast having the ability to produce lactic acid may retain the LDH in a plasmid or YAC maintained outside the yeast chromosome, but as described above, it may be incorporated and retained in the yeast chromosome. preferable. Although there is no restriction | limiting in particular as a method to introduce | transduce LDH into a yeast chromosome, For example, it can introduce | transduce by the method disclosed by Unexamined-Japanese-Patent No. 2008-29329. Further, in the higher polyploid yeast having the ability to produce lactic acid, LDH is retained at least 1, preferably 2 or more, more preferably 3 or more, and further preferably 4 or more.

  The fermentation raw material used in the present invention is not limited as long as it promotes the growth of yeast to be cultured and can produce lactic acid as a target fermentation product satisfactorily. For example, a carbon source, a nitrogen source, inorganic salts, A liquid medium or the like suitably containing organic micronutrients such as amino acids and vitamins as needed is preferably used. Examples of the carbon source include sugars such as glucose, sucrose, fructose, galactose, lactose and maltose, starch saccharified liquid containing these sugars, sweet potato molasses, sugar beet molasses, high test molasses, cane juice, and cane juice extraction Product or concentrate, raw sugar purified or crystallized from cane juice, purified sugar crystallized or crystallized from cane juice, organic acids such as acetic acid and fumaric acid, alcohols such as ethanol, and glycerin Etc. are preferably used. Sugars are the first oxidation products of polyhydric alcohols, and are carbohydrates that have one aldehyde group or ketone group, sugars with aldehyde groups are classified as aldoses, and sugars with ketone groups are classified as ketoses. It is preferably glucose, sucrose, fructose, galactose, lactose or maltose. The above carbon source may be added all at once at the start of the culture, or may be added in portions during the culture or continuously.

  Examples of the nitrogen source include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein decomposition products, Other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used.

  Moreover, as said inorganic salt, a phosphate, magnesium salt, calcium salt, iron salt, manganese salt etc. can be suitably added and used, for example.

  Moreover, when the higher polyploid yeast having the ability to produce lactic acid is auxotrophic, the nutrient can be added as a preparation or a natural product containing it. An antifoaming agent can be added and used as necessary.

  Although there is no particular limitation on the fermentation culture conditions of the higher-order polyploid yeast having the ability to produce lactic acid as long as it can be cultured, it is preferably carried out at a pH of 4 to 8 and a temperature of 20 to 40 ° C. The pH of the fermentation broth is adjusted to a predetermined value within the above range with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, and the like.

  In fermentation culture of higher-order polyploid yeast having the ability to produce lactic acid, if it is necessary to increase the oxygen supply rate, oxygen is added to the air to keep the oxygen concentration at 21% or higher, the culture solution is pressurized, and stirring Means such as increasing the speed or increasing the air flow rate can be used. Conversely, if it is necessary to reduce the oxygen supply rate, it is also possible to supply a gas containing no oxygen such as carbon dioxide, nitrogen and argon mixed with air.

  In the present invention, batch culture or fed-batch culture may be performed at the initial stage of culture to increase the bacterial cell concentration, and then continuous culture (pulling) may be started. Continuous culture may be performed. It is possible to supply the fermentation raw material liquid and extract the culture from an appropriate time. The start time of the fermentation raw material supply and the withdrawal of the culture are not necessarily the same. Further, the supply of the fermentation raw material liquid and the withdrawal of the culture may be continuous or intermittent. What is necessary is just to add a nutrient required for microbial cell growth as shown above to a fermentation raw material liquid so that microbial cell growth may be performed continuously.

  In order to obtain efficient productivity, it is necessary to maintain the cell concentration in the fermentation broth at a high level so long as the ratio of the environment of the fermentation broth is inappropriate for the growth of the cell and the rate of death does not increase. As an example, good production efficiency can be obtained by maintaining the dry weight at 5 g / L or more. Moreover, the upper limit of the density | concentration of a microbial cell is not specifically limited unless the malfunction on the operation of a continuous fermentation apparatus or the fall of production efficiency is caused.

  The continuous culture operation performed while growing fresh cells having fermentation production ability is usually preferably performed in a single fermentation reaction tank in terms of culture management. However, the number of fermentation reaction tanks is not limited as long as it is a continuous culture method for producing a product while growing cells. A plurality of fermentation reaction tanks may be used because the capacity of the fermentation reaction tank is small. In this case, high productivity of the fermentation product can be obtained even if continuous fermentation is performed by connecting a plurality of fermentation reaction tanks in parallel or in series by piping.

In the present invention, a continuous fermentation continues, illustrating a state in which withdrawal of the culture with the supply of the fermentation raw material liquid is continuously or during are missing performed. In the present invention, it is preferable that lactic acid is efficiently produced during the continuous fermentation. Lactic acid is efficiently produced as evaluated by lactic acid accumulation concentration, lactic acid production rate, lactic acid vs. sugar yield, and any one, preferably two, and more preferably all are in a high state. That is.

  The lactic acid accumulation concentration is the concentration of lactic acid contained in the culture solution, and the state where the lactic acid accumulation concentration is high is 40 g / L or more, preferably 42 g / L, more preferably 44 g / L.

  The lactic acid production rate is the amount of lactic acid produced per unit time, and the state where the lactic acid production rate is high means that the lactic acid production rate represented by Formula 3 is 7.5 g / L / h or more, preferably 8 or more. More preferably, it is 9 g / L / h or more.

  Lactic acid-to-sugar yield is the ratio of the amount of lactic acid produced from the carbon source consumed per unit time, and the high lactic acid-to-sugar yield is the lactic acid-to-sugar yield expressed by Equation 4. The rate is 70% or more, preferably 75% or more, more preferably 80% or more.

  Separation / purification of lactic acid contained in the filtered / separated fermentation broth produced according to the present invention can be performed by combining conventionally known methods such as concentration, distillation and crystallization. For example, filtration / separated fermented broth Extraction with diethyl ether or ethyl acetate after reducing the pH of the solution to 1 or less, a method of elution after adsorbing and washing with an ion exchange resin, a method of reacting with alcohol in the presence of an acid catalyst and distilling as an ester, and calcium Examples thereof include a method of crystallizing as a salt or a lithium salt, and a separation / purification method combining nanofiltration membrane and distillation disclosed in WO2009 / 004922.

  Next, the preferable aspect of the continuous fermentation apparatus used with the manufacturing method of lactic acid of this invention is demonstrated using figures.

  FIG. 1 is a schematic side view for explaining a preferred example of a membrane separation type continuous fermentation apparatus used in the method for producing lactic acid by continuous fermentation of the present invention. FIG. 1 is a typical example in which the separation membrane element is installed outside the fermentation reaction tank.

In FIG. 1, a membrane separation type continuous fermentation apparatus basically includes a fermentation reaction tank 1, a membrane separation tank 12, and a differential pressure control device 3. Here, the separation membrane element 2 incorporates a porous separation membrane. As the porous separation membrane, for example, a separation membrane and a separation membrane element disclosed in International Publication No. 2002/064240 are suitably used. The membrane separation tank 12 is connected to the fermentation reaction tank 1 via a fermentation culture medium circulation pump 11.

  In FIG. 1, a culture medium is put into the fermentation reaction tank 1 by a medium supply pump 7, the fermentation culture solution in the fermentation reaction tank 1 is stirred with a stirrer 5 as necessary, and a gas supply device 4 is used as necessary. Can supply the required gas. At this time, the supplied gas can be recovered and recycled and supplied again by the gas supply device 4. If necessary, the pH of the fermentation broth is adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8, and the temperature of the fermentation broth is adjusted by the temperature controller 10 as necessary. Fermentative production with high productivity can be performed. Furthermore, the fermentation liquid in the apparatus is circulated between the fermentation reaction tank 1 and the membrane separation tank 12 by the fermentation liquid circulation pump 11. The fermentation broth containing the fermentation product is filtered and separated into microorganisms and fermentation product by the separation membrane element 2 and can be taken out from the apparatus system.

  Moreover, the microorganisms filtered and separated remain in the apparatus system, so that the microorganism concentration in the apparatus system can be maintained high, and fermentation production with high productivity is possible. Here, the filtration / separation by the separation membrane element 2 can be performed without using any special power due to the water head differential pressure with respect to the water surface of the membrane separation tank 12. The pressure control device 3 can appropriately adjust the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation broth in the device system. If necessary, the required gas can be supplied into the membrane separation tank 12 by the gas supply device 4. At this time, the supplied gas can be recovered and recycled and supplied again by the gas supply device 4. The filtration / separation by the separation membrane element 2 can be performed by suction filtration using a pump or the like, or by pressurizing the inside of the apparatus system as necessary. Moreover, a high-order polyploid yeast can be cultured for continuous fermentation in a culture tank, and can be supplied into the fermentation tank as necessary. By culturing higher-order polyploid yeast in a culture tank and supplying it to the fermenter as needed, continuous fermentation with fresh and high-order polyploid yeast with a high production capacity for lactic acid becomes possible, and high production performance is achieved. Continuous fermentation maintained for a long time is possible.

  Next, FIG. 2 is a schematic side view for explaining an example of another membrane separation type continuous fermentation apparatus used in the present invention. Of the continuous fermentation apparatus used in the method for producing lactic acid of the present invention, a typical example in which a separation membrane element is installed inside a fermentation reaction tank is shown in the schematic diagram of FIG.

  In FIG. 2, the membrane separation type continuous fermentation apparatus basically includes a fermentation reaction tank 1 and a differential pressure control apparatus 3. A porous membrane is incorporated in the separation membrane element 2 in the fermentation reaction tank 1. As this porous separation membrane, for example, a separation membrane and a separation membrane element disclosed in WO2002 / 064240 can be used. The separation membrane element will be described in detail later.

  Next, the form of continuous fermentation by the membrane separation type continuous fermentation apparatus of FIG. 2 will be described. The culture medium is pumped continuously or intermittently into the fermentation reaction tank 1 by the culture medium supply pump 7. The medium can be sterilized by heat sterilization, heat sterilization, or sterilization using a filter as necessary before charging. At the time of fermentation production, the fermentation culture solution in the fermentation reaction tank 1 is agitated by the agitator 5 in the fermentation reaction tank 1 as necessary. At the time of fermentation production, the gas required by the gas supply device 4 can be supplied into the fermentation reaction tank 1 as necessary. During fermentation production, if necessary, the pH of the fermentation broth in the fermentation reaction tank 1 is adjusted by the pH sensor / control device 9 and the pH adjustment solution supply pump 8, and the fermentation is performed by the temperature controller 10 as necessary. By adjusting the temperature of the fermentation broth in the reaction tank 1, fermentation production with high productivity can be performed. Here, the pH and temperature are exemplified for the adjustment of the physicochemical conditions of the fermentation broth by the instrumentation / control device. However, if necessary, the dissolved oxygen and ORP are controlled by an analyzer such as an online chemical sensor, The concentration of lactic acid in the fermentation broth can be measured, and the physicochemical conditions can be controlled using the concentration of lactic acid in the fermentation broth as an index. In addition, the continuous or intermittent input of the medium is preferably performed by appropriately adjusting the amount and speed of the medium input using the measured value of the physicochemical environment of the fermentation broth by the instrumentation apparatus as an index.

  In FIG. 2, the fermentation broth is filtered and separated by the separation membrane element 2 installed in the fermentation reaction tank 1, and the fermentation product is taken out from the apparatus system. In addition, since the filtered and separated cells remain in the apparatus system, the concentration of the cells in the apparatus system can be maintained high, and fermentation production with high productivity is possible. Here, the filtration / separation by the separation membrane element 2 is performed by the water head differential pressure with respect to the water surface of the fermentation reaction tank 1 and can be performed without using any special power. The pressure control device 3 can appropriately adjust the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation broth in the fermentation reaction tank 1. For the filtration / separation by the separation membrane element, filtration / separation can be performed by suction filtration using a pump or the like, or pressurizing the inside of the apparatus system, if necessary. Moreover, a microbial cell can be cultured with the culture tank prepared separately, and can be supplied in a fermenter as needed. By culturing the microbial cells in a culture tank and supplying them into the fermenter as necessary, continuous fermentation with fresh microbial cells is always possible, and continuous fermentation with high lactic acid production performance maintained for a long period of time becomes possible.

  The separation membrane element preferably used in the continuous fermentation apparatus used in the method for producing lactic acid according to the present invention will be described with reference to the drawings. In the continuous fermentation apparatus used in the method for producing lactic acid according to the present invention, the separation membrane and the separation membrane element disclosed in International Publication No. 2002/064240 can be preferably used.

  The form of the separation membrane element used in the present invention will be outlined below with reference to the drawings of the separation membrane and the separation membrane element disclosed in WO 2002/064240, which is an example of a preferred embodiment. To do. FIG. 3 is a schematic perspective view for explaining one embodiment of the separation membrane element used in the present invention.

  As shown in FIG. 3, the separation membrane element is configured by arranging the flow path material 14 and the separation membrane 15 in this order on both surfaces of a rigid support plate 13. The support plate 13 has recesses 16 on both sides. The separation membrane 15 filters the fermentation broth. The flow path member 14 is for efficiently flowing the permeated water filtered by the separation membrane 15 to the support plate 13. The permeated water that has flowed to the support plate 13 passes through the recess 16 of the support plate 13 and is taken out of the continuous fermentation apparatus via a water collecting pipe 17 that is a discharge means.

  Next, another embodiment of the separation membrane element shown in FIG. 4 will be described. FIG. 4 is a cross-sectional explanatory diagram for explaining an example of another separation membrane element used in the present invention.

Separation membrane element, as shown in FIG. 4, the separation membrane bundle composed of hollow fiber membranes 18 are adhered and fixed to the upper parts of the resin sealing layer 19 and the lower resin sealing layer 20 thus bundle. Adhesion / fixation by the lower resin sealing layer 20 seals the hollow portion of the hollow fiber membrane and has a structure that prevents leakage of the fermentation broth. On the other hand, the upper resin sealing layer 19 does not seal the inner hole of the hollow fiber membrane, and has a structure in which permeate flows through the water collecting pipe 22. This separation membrane element can be installed in the continuous fermentation apparatus via the support frame 21. The permeated water filtered by the separation membrane bundle 18 passes through the hollow portion of the hollow fiber membrane and is taken out of the fermentation culture tank via the water collecting pipe 22. As power for taking out the permeated water, a method such as a water head differential pressure, a pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used.

  The member constituting the separation membrane element of the continuous fermentation apparatus used in the method for producing lactic acid by continuous fermentation of the present invention is preferably a member resistant to high-pressure steam sterilization operation. If the inside of the fermentation apparatus can be sterilized, it is possible to avoid the risk of contamination with undesirable cells during continuous fermentation, and more stable continuous fermentation is possible. The member constituting the separation membrane element is preferably resistant to the treatment for 15 minutes at a temperature of 121 ° C., which is the condition of the high-pressure steam sterilization operation. Examples of separation membrane element members include metals such as stainless steel and aluminum, polyamide resins, fluorine resins, polycarbonate resins, polyacetal resins, polybutylene terephthalate resins, PVDF, modified polyphenylene ether resins, and polysulfone resins. These resins can be preferably selected.

  In the continuous fermentation apparatus used in the method for producing lactic acid by continuous fermentation of the present invention, the separation membrane element may be installed outside the fermentation tank or may be installed in the fermentation reaction tank. When the separation membrane element is installed outside the fermentation reaction tank, a separate membrane separation tank can be provided and the separation membrane element can be installed inside it, and the fermentation broth is circulated between the fermentation reaction tank and the membrane separation tank. The fermentation broth can be continuously filtered by the separation membrane element.

  In the continuous fermentation apparatus used in the method for producing lactic acid by continuous fermentation according to the present invention, the membrane separation tank is preferably capable of high-pressure steam sterilization. By making the membrane separation tank capable of high-pressure steam sterilization, it is easy to avoid contamination by various bacteria.

  Hereinafter, the present invention will be described using examples.

(Reference Example 1) Production of a higher-order polyploid yeast having the ability to produce lactic acid It is obtained by introducing an L-lactate dehydrogenase gene derived from Xenopus (hereinafter also referred to as XLDH) into Saccharomyces cerevisiae NBRC10505 strain. A high-order polyploid yeast having the ability to produce lactic acid was prepared by making a yeast having the ability to produce lactic acid into a higher-order polyploid. Furthermore, as described in detail below, restoration of auxotrophy and the property of temperature-sensitive mutation were imparted. Table 2 shows the yeasts used for preparing the higher-order polyploid yeast and yeasts prepared by the method described below. Hereinafter, detailed procedures for LDH insertion, temperature-sensitive mutation, and auxotrophic recovery methods will be described.

(No. 1) Construction of Xenopus L-LDH (XLDH) Expression Vector Cloning of XLDH (SEQ ID NO: 1) was performed by PCR. For PCR, phagemid DNA prepared from Xenopus kidney cDNA library (manufactured by STRATAGENE) according to the attached protocol was used as a template.

For the PCR amplification reaction, KOD-Plus polymerase (manufactured by Toyobo Co., Ltd.) was used, and the attached reaction buffer, dNTPmix, etc. were used. Phagemid DNA was made adjustment follow the attached protocol as described above 50ng / sample, were prepared in the reaction system of 50μl so 50 pmol sample primers, and KOD-Plus polymerase 1 unit / sample. The reaction solution was thermally denatured at 94 ° C. for 5 minutes with a PCR amplification apparatus iCycler (manufactured by BIO-RAD), then 94 ° C. (thermal denature): 30 seconds, 55 ° C. (primer annealing): 30 seconds, 68 C. (Complementary strand extension): One cycle was performed for 30 cycles, and then cooled to a temperature of 4.degree. The gene amplification primer (SEQ ID NO: 2 and 3) was prepared such that a SalI recognition sequence was added to the 5 terminal side and a NotI recognition sequence was added to the 3 terminal side.

  The PCR amplified fragment was purified, the end was phosphorylated with T4 polynucleotide Kinase (manufactured by Takara Bio Inc.), and then ligated to the pUC118 vector (cut with the restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver.2 (Takara Bio Inc.). The ligation solution was transformed into competent cells of Escherichia coli DH5α (manufactured by Takara Bio Inc.) and plated on an LB plate containing 50 μg / mL of the antibiotic ampicillin and cultured overnight. About the grown colonies, plasmid DNA was collected with a miniprep, cut with restriction enzymes SalI and NotI, and a plasmid in which the Xenopus laevis LDH gene was inserted was selected. All of these series of operations were performed according to the attached protocol.

  The pUC118 vector into which XLDH was inserted was cleaved with restriction enzymes SalI and NotI, the DNA fragments were separated by 1% agarose gel electrophoresis, and the fragments containing XLDH were purified according to a conventional method. The obtained fragment was ligated to the XhoI / NotI cleavage site of the expression vector pTRS11 shown in FIG. 5, and plasmid DNA was recovered in the same manner as described above, and then cut with the restriction enzymes XhoI and NotI to insert XLDH. The expression vector pTRS102 was selected.

  A method for inserting LDH into the PDC1, SED1, and TDH3 gene loci using pTRS102 obtained as described above as a template for XLDH will be described below.

(Part 2) Insertion of LDH into the yeast chromosome PDC1 locus A 1.3-kb PCR fragment containing XLDH and TDH3 terminator sequences by PCR using pTRS102 as an amplification template and oligonucleotides (SEQ ID NOs: 4 and 5) as primer sets Was amplified. Here, SEQ ID NO: 4 was designed such that a sequence corresponding to 60 bp upstream from the start codon of the PDC1 gene was added.

Next, a 1.2 kb PCR fragment containing the TRP1 gene as a yeast selection marker was amplified by PCR using the plasmid pTRS424 as an amplification template and oligonucleotides (SEQ ID NOs: 6 and 7) as primer sets. Here, SEQ ID NO: 7 was designed to the sequence corresponding to the downstream 60bp from the end stop codon of PDC1 gene are added.

  Each DNA fragment was separated by 1% agarose gel electrophoresis and purified according to a conventional method. A mixture of each 1.3 kb fragment and 1.2 kb fragment obtained as described above was used as an amplification template, and PCR was performed using oligonucleotides (SEQ ID NOs: 4 and 7) as primer sets. A PCR fragment of about 2.5 kb to which XLDH, TDH3 terminator, and TRP1 gene were ligated, to which sequences corresponding to 60 bp upstream and downstream of the gene were added, was amplified.

  The above PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, transformed, and cultured in a medium without tryptophan, so that XLDH was introduced downstream of the PDC1 gene promoter on the chromosome. Transformants were selected.

  Confirmation that the transformant obtained as described above was a yeast in which Xenopus L-LDH was introduced downstream of the PDC1 gene promoter on the chromosome was performed as follows. First, the genomic DNA of the transformant was prepared using a genomic DNA extraction kit Gen Toru-kun (manufactured by Takara Bio Inc.), and this was used as an amplification template, and by PCR using oligonucleotides (SEQ ID NOs: 7 and 8) as primer sets, This was confirmed by obtaining an amplified DNA fragment of 2.8 kb. For non-transformed strains, an amplified DNA fragment of about 2.1 kb can be obtained by the PCR.

(Part 3) Insertion of LDH into the yeast chromosome SED1 locus The introduction into the SED1 locus was performed by PCR using the pTRS102 prepared in Reference Example 1 as an amplification template and oligonucleotides (SEQ ID NOs: 5 and 9) as primer sets. A 1.3 kb PCR fragment containing the LDH gene derived from Xenopus laevis and the TDH3 terminator sequence was amplified. Here, SEQ ID NO: 9 was designed such that a sequence corresponding to 60 bp upstream from the start codon of the SED1 gene was added.

Next, a PCR fragment of about 1.3 kb containing the HIS3 gene, which is a yeast selection marker, was amplified by PCR using plasmid pRS423 as an amplification template and oligonucleotides (SEQ ID NOs: 6, 10) as primer sets. Here, SEQ ID NO: 10 was designed to the sequence corresponding to the downstream 60bp from the end stop codon of SED1 gene is added.

  Each DNA fragment was separated by 1% agarose gel electrophoresis and purified according to a conventional method. A mixture of the two kinds of about 1.3 kb fragments obtained here was used as an amplification template, and PCR was performed using oligonucleotides (SEQ ID NOs: 9 and 10) as primer sets. An about 2.6 kb PCR fragment to which a sequence corresponding to 60 bp upstream / downstream was added and a LDH gene derived from Xenopus laevis, a TDH3 terminator, and a HIS3 gene were ligated was amplified.

  The above PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, transformed, and cultured in a medium without histidine, so that XLDH was introduced downstream of the SED1 gene promoter on the chromosome. Transformants were selected.

  Confirmation that the transformed strain obtained as described above was a yeast in which XLDH was introduced downstream of the SED1 gene promoter on the chromosome was performed as follows. First, genomic DNA of the transformed strain was prepared by a genomic DNA extraction kit Gen Toru-kun (manufactured by Takara Bio Inc.), and this was used as an amplification template, and by PCR using oligonucleotides (SEQ ID NOs: 11 and 12) as primer sets, This was confirmed by obtaining an amplified DNA fragment of 2.9 kb. For non-transformed strains, an amplified DNA fragment of about 1.4 kb can be obtained by the PCR.

(Part 4) Introduction of LDH into the yeast chromosome TDH3 locus For introduction into the TDH3 locus, a plasmid was prepared in which the TDH3 terminator of pTRS102 was changed to the ADH1 terminator.

  First, genomic DNA was extracted from the NBRC10505 strain using a genomic DNA extraction kit “Gen Tor-kun” (manufactured by Takara Bio Inc.), PCR using the extracted genomic DNA as a template and oligonucleotides (SEQ ID NOs: 13 and 14) as primer sets The PCR fragment containing the ADH1 promoter was amplified. Here, SEQ ID NO: 13 was prepared such that a NotI recognition sequence was added to the 5 terminal side, and a HindIII recognition sequence was added to SEQ ID NO: 14 on the 3 terminal side.

  The PCR amplified fragment was purified, the end was phosphorylated with T4 polynucleotide Kinase (manufactured by Takara Bio Inc.), and then ligated to the pUC118 vector (cut with the restriction enzyme HincII and the cut surface was dephosphorylated). The ligation solution was transformed into competent cells of Escherichia coli DH5α (manufactured by Takara Bio Inc.) and plated on an LB plate containing 50 μg / mL of the antibiotic ampicillin and cultured overnight. For the grown colonies, plasmid DNA was collected with a miniprep, cut with restriction enzymes NotI and HindIII, and a plasmid in which the ADH1 terminator was inserted was selected. The prepared plasmid is designated as pUC118-ADH1t.

  Next, pUC118-ADH1t was cleaved with restriction enzymes NotI and HindIII, DNA fragments were separated by 1% agarose gel electrophoresis, and a fragment containing ADH1 terminator was purified according to a routine method. The obtained fragment containing the ADH1 terminator was ligated to the NotI / HindIII cleavage site of pTRS102, and plasmid DNA was recovered by the same method as described above, and then cleaved with the restriction enzymes NotI and HindIII. Modified plasmids were selected. Hereinafter, the plasmid thus prepared is designated as pTRS150.

  A 1.3 kb PCR fragment containing the frog-derived L-LDH gene and the ADH1 terminator sequence was amplified by PCR using the pTRS150 as a template and oligonucleotides (SEQ ID NOs: 15 and 16) as primer sets. Here, the primer of SEQ ID NO: 16 was designed such that a sequence corresponding to 60 bp upstream from the start codon of the TDH3 gene was added.

Next, a 1.2 kb PCR fragment containing the URA3 gene, a yeast selection marker, was amplified by PCR using plasmid pRS426 as an amplification template and oligonucleotides (SEQ ID NOs: 17 and 18) as primer sets. Here, the primer of SEQ ID NO: 18, was designed to the sequence corresponding to the downstream 60bp from the end stop codon of the TDH3 gene is added.

  Each PCR fragment was separated by 1% agarose gel electrophoresis and purified according to a conventional method. A PCR method using the mixture of each 1.3 kb fragment and 1.2 kb fragment obtained here as an amplification template and oligonucleotides (SEQ ID NOs: 16 and 18) as primer sets was used to detect XLDH, ADH1 terminator and URA3 gene. The ligated PCR fragment of about 2.5 kb was amplified.

  The above PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, transformed, and cultured in a medium without uracil, so that XLDH was introduced downstream of the TDH3 gene promoter on the chromosome. Transformants were selected.

  Confirmation that the transformant obtained as described above was a yeast in which XLDH was introduced downstream of the TDH3 gene promoter on the chromosome was performed as follows. First, genomic DNA of the transformed strain was prepared by a genomic DNA extraction kit “Gen Tor-kun” (manufactured by Takara Bio Inc.), which was used as an amplification template and by PCR using oligonucleotides (SEQ ID NOs: 19 and 20) as primer sets. It was confirmed that an amplified DNA fragment of about 2.8 kb was obtained. For non-transformed strains, an amplified DNA fragment of about 2.1 kb can be obtained by the PCR.

(Part 5) Obtaining temperature-sensitive mutation of yeast having a PDC5 gene having a temperature-sensitive mutation The yeast SW015 strain having a temperature-sensitive mutation in the pdc5 gene described in JP-A-2008-048726, and temperature sensitivity It was obtained by conjugating with the yeast to be mutated. The diploid yeast is ascended with an ascending medium, dissected with a micromanipulator, each haploid cell is obtained, and the auxotrophy of each haploid cell is examined, From the obtained haploid cells, a strain having XLDH inserted into any of the PDC1 gene, SED1 gene, and TDH1 locus and having a temperature-sensitive mutation in the PDC5 gene (incapable of growing at 34 ° C.) was selected.
Next, we will explain how to restore nutritional requirements.

(6) Preparation of lysine auxotrophic return strain To restore lysine auxotrophy, the following method was used. A PCR fragment of about 2 kb in the first half of the LYS2 gene was amplified by PCR using BY4741 genomic DNA manufactured by Funakoshi as a template and oligonucleotides (SEQ ID NOS: 21 and 22) as primer sets. The PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, and then subjected to a transformation operation to release the amber mutation of the LYS2 gene. By culturing in a lysine-free medium, a transformant having restored lysine synthesis ability was selected.

  Confirmation that the transformant obtained as described above was a yeast from which the amber mutation of the LYS2 gene was released was performed as follows. First, the obtained transformant and 20GY77 strain having a wild-type LYS2 gene were joined to obtain diploid cells. The diploid cells were allowed to form ascending with an ascending medium. The ascending sac was dissected with a micromanipulator to obtain each haploid cell, and the auxotrophy of each haploid cell was examined. All of the obtained haploid cells were confirmed to have lysine synthesis ability. When the lysine synthesis ability is restored without releasing the mutation of the LYS2 gene, cells having no lysine synthesis ability are obtained from the haploid cells obtained above.

(Part 7) Acquisition of leucine auxotrophic return strain To restore leucine auxotrophy, the following method was used. About 2 kb of the PCR fragment of the LEU2 gene was amplified by PCR using the plasmid PRS425 as a template and oligonucleotides (SEQ ID NOs: 23, 24) as primer sets. The above PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, and then subjected to a transformation operation to release the mutation of the LEU2 gene. By culturing in a leucine-free medium, a transformant in which the ability to synthesize leucine was restored was selected.

  Confirmation that the transformant obtained as described above was a yeast in which the mutation of the LEU2 gene was released was performed as follows. First, the obtained transformant was joined to Saccharomyces cerevisiae 20GY7 strain having a wild type LEU2 gene to obtain diploid cells. The diploid cells were allowed to form ascending with an ascending medium. The ascending sac was dissected with a micromanipulator to obtain each haploid cell, and the auxotrophy of each haploid cell was examined. All of the obtained haploid cells were confirmed to have the ability to synthesize leucine. In addition, when the leucine synthesis ability is restored without canceling the mutation of the LEU2 gene, cells having no leucine synthesis ability are obtained among the haploid cells obtained above.

(No. 8) Acquisition of adenine auxotrophic return strain In order to restore adenine auxotrophy, the following method was used. About 2 kb of the PCR fragment of the ADE2 gene was amplified by PCR using the plasmid PRS422 as a template and oligonucleotides (SEQ ID NOs: 25 and 26) as primer sets. Separated by 1% agarose gel electrophoresis of the PCR fragment described above, after purification by a conventional method, and the cells operation, releasing the mutation of ADE 2 gene. By culturing in adenine-free medium, were selected transformants adenine case forming ability is restored.

  Confirmation that the transformant obtained as described above was a yeast in which the mutation of the AED2 gene was canceled was performed as follows. First, diploid cells were obtained by joining the obtained transformant to Saccharomyces cerevisiae 20GY7 strain having a wild-type ADE2 gene. The diploid cells were allowed to form ascending with an ascending medium. The ascending sac was dissected with a micromanipulator to obtain each haploid cell, and the auxotrophy of each haploid cell was examined. It was confirmed that all of the obtained haploid cells had the ability to synthesize adenine. When the ADE2 gene mutation is not released and adenine synthesis ability is restored, cells having no adenine synthesis ability are obtained from the haploid cells obtained above.

(No. 9) Acquisition of uracil auxotrophic return strain To restore uracil auxotrophy, the following method was used. About 2 kb of PCR fragment of URA3 gene was amplified by PCR using plasmid pRS426 as a template and oligonucleotide (SEQ ID NO: 27, 28) as a primer set. The PCR fragment was separated by 1% agarose gel electrophoresis, purified according to a conventional method, and then subjected to a transformation operation to release the mutation in the URA3 gene. By culturing in a medium not containing uracil, a transformant in which uracil synthesis ability was restored was selected.

  Confirmation that the transformant obtained as described above was a yeast in which the mutation of the URA3 gene was released was performed as follows. First, the obtained transformant and Saccharomyces cerevisiae 20GY7 strain having a wild-type URA3 gene were joined to obtain diploid cells. The diploid cells were allowed to form ascending with an ascending medium. The ascending sac was dissected with a micromanipulator to obtain each haploid cell, and the auxotrophy of each haploid cell was examined. All of the obtained haploid cells were confirmed to have uracil synthesis ability. In addition, when the URA3 gene mutation is not released and uracil synthesis ability is restored, cells having no uracil synthesis ability are obtained from the haploid cells obtained above.

(No. 10) Acquisition of a higher-order polyploid strain having the ability to produce lactic acid. A transformant obtained by combining the methods 1 to 9 is joined to obtain a diploid prototrophic strain having no auxotrophy. It was. Furthermore, the diploid prototrophic strain was made to form ascosm in an ascending medium, and the ascend was dissected with a micromanipulator to obtain each haploid cell. The obtained haploid cells were examined for auxotrophy, and the strains grown on the SD medium (Table 1) were judged to have returned to auxotrophy, and designated as haploid prototrophs.

(Reference Example 2) Quantification Method of Lactic Acid Lactic acid was confirmed by measuring the amount of lactic acid by the HPLC method under the following conditions for the centrifugal supernatant of the culture solution.

Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
Detection method: electrical conductivity Temperature: 45 ° C.

  The optical purity of L-lactic acid was measured by the HPLC method under the following conditions.

Column: TSK-gel Enantio L1 (manufactured by Tosoh Corporation)
Mobile phase: 1 mM aqueous copper sulfate flow rate: 1.0 ml / min
Detection method: UV254 nm
Temperature: 30 ° C.

Moreover, the optical purity of L-lactic acid was calculated by the following formula.
Optical purity (%) = 100 × (LD) / (L + D)
Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid.

(Reference Example 3) Production of porous membrane (Part 1)
Polyvinylidene fluoride (PVDF) resin was used as the resin and N, N-dimethylacetamide (DMAc) was used as the solvent, and these were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
[Undiluted solution]
・ PVDF: 13.0% by weight
DMAc: 87.0% by weight.

Next, after the above-mentioned stock solution was cooled to a temperature of 25 ° C., a non-woven fabric made of polyester fiber having a density of 0.48 g / cm ³ and a thickness of 220 μm (porous base) And immediately immersed in a coagulation bath at 25 ° C. having the following composition for 5 minutes to obtain a flat film having a porous resin layer formed on a porous substrate.
[Coagulation bath]
-Water: 30.0% by weight
DMAc: 70.0% by weight.

After peeling this porous membrane from the glass plate, it was immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc to obtain a separation membrane. When the surface of the porous resin layer was observed with a scanning electron microscope at a magnification of 10,000 within the range of 9.2 μm × 10.4 μm, the average diameter of all observable pores was 0.1 μm. . Next, when the pure water permeability coefficient was evaluated about the said separation membrane, it was 50 * 10 < ^-9 > m < ³ > / m < ² > / s / Pa. The standard deviation of the average pore diameter was 0.035 μm, and the membrane surface roughness was 0.06 μm.

(Reference Example 4) Production of porous membrane (part 2)
A vinylidene fluoride homopolymer having a weight average molecular weight of 41,000 and γ-butyrolactone were dissolved at a temperature of 170 ° C. at a ratio of 38% by weight and 62% by weight, respectively, to prepare a stock solution. This stock solution was discharged from the base while stirring γ-butyrolactone as a hollow portion forming liquid, and solidified in a cooling bath composed of an 80% by weight aqueous solution of γ-butyrolactone at a temperature of 20 ° C. to produce a hollow fiber membrane.

Next, 14% by weight of vinylidene fluoride homopolymer having a weight average molecular weight of 284,000, 1% by weight of cellulose acetate propionate (Eastman Chemical Co., CAP482-0.5), and N-methyl-2-pyrrolidone 77% by weight, polyoxyethylene coconut oil fatty acid sorbitan (manufactured by Sanyo Chemical Co., Ltd., trade name “IONET T-20C” (registered trademark)) 5% by weight, and water 3% by weight at a temperature of 95 ° C. mixed and dissolved to, was made by adjusting the stock. This undiluted solution was uniformly applied to the surface of the hollow fiber membrane obtained above, and a hollow fiber membrane (porous membrane) used in the present invention was immediately solidified in a water bath. The average pore diameter of the treated water side surface of the obtained hollow fiber membrane (separation membrane) was 0.05 μm. Next, when the pure water permeability coefficient was evaluated about the hollow fiber membrane which is said separation membrane, it was 5.5 * 10 < ^-9 > m < ³ > / m < ² > * s * Pa. The standard deviation of the average pore diameter was 0.006 μm.

(Example 1) Production of lactic acid by continuous fermentation using a high-order polyploid (nutrient non-requirement) yeast having the ability to produce lactic acid (Part 1)
Using the yeast HI003 strain prepared in Reference Example 1, lactic acid was produced using the continuous fermentation apparatus of FIG. 1 and the SC4 medium having the composition shown in Table 3. The medium was used after autoclaving at 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, the porous membrane prepared in Reference Example 3 was used.

The operating conditions in Example 1 are as follows.
・ Fermentation reactor capacity: 2 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 3)
・ Membrane separation element effective filtration area: 60cm2
・ Temperature adjustment: 32 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
-Aeration rate of membrane separation tank: 0.3 (L / min)
・ Fermentation reactor stirring speed: 100 (rpm)
-PH adjustment: pH 5 adjusted with 8N Ca (OH) 2-Lactic acid fermentation medium supply rate: 50-300 ml / hr. In the range of circulated fluid volume by variable control and fermentation fluid circulation device: 0.1 (L / min)
-Membrane permeate flow rate control: Flow rate control by transmembrane differential pressure (transmembrane differential pressure: controlled at 0.1 kPa to less than 20 kPa)
-The culture tank containing the separation membrane element is autoclaved at 121 ° C for 20 min and autoclaved.

  The HPLC shown in Reference Example 2 was used for evaluation of the concentration of lactic acid as a product, and “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used for the measurement of glucose concentration. It was.

  First, HI003 strain was shaken overnight in 5 ml of SC4 medium in a test tube to obtain a culture solution (pre-culture). The obtained culture solution was inoculated into 100 ml of fresh SC4 medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The incubating medium is inoculated in a 1.5 L SC4 medium of the continuous fermentation apparatus shown in FIG. 1, and the fermentation reaction tank 1 is stirred by the attached stirrer 5 to adjust the aeration rate of the fermentation reaction tank 1 and adjust the temperature. Adjustment and pH adjustment were carried out for 24 hours without operating the fermentation broth circulation pump 11 (pre-culture). Immediately after completion of the pre-culture, the fermentation broth circulation pump 11 is operated, in addition to the operating conditions during pre-culture, the membrane separation tank 12 is vented, the SC4 medium is continuously supplied, and the fermentation of the membrane separation type continuous fermentation apparatus Continuous culture was performed while controlling the amount of membrane permeated water so that the amount of the culture solution was 2 L, and lactic acid was produced by continuous fermentation. Control of the amount of permeated water through the continuous fermentation test was performed by appropriately changing the transmembrane differential pressure to be 0.1 kPa or more and less than 20 kPa by the differential pressure control device 3. The produced lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately.

  As a result of the 800-hour continuous fermentation test, stable lactic acid production by continuous fermentation was possible as shown in Table 6.

(Example 2) Production of lactic acid by continuous fermentation using a higher-order polyploid (nutrient non-requirement) yeast having the ability to produce lactic acid (part 2)
Using the yeast HI003 strain prepared in Reference Example 1, lactic acid was produced using the continuous fermentation apparatus shown in FIG. 2 and the SC4 medium having the composition shown in Table 3. The medium was used after autoclaving at 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, the porous separation membrane prepared in Reference Example 3 was used.

The operating conditions in Example 2 are as follows.
・ Fermentation reactor capacity: 2 (L)
-Separation membrane used: Polyvinylidene fluoride filtration membrane (Reference Example 3)
・ Membrane separation element effective filtration area: 120 cm ²
・ Temperature adjustment: 32 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
Lactic acid fermentation medium supply rate: 50 to 300 ml / hr. Variable control within the range of-Stirring speed of fermentation reaction tank: 800 (rpm)
・ PH adjustment: Adjust pH to 5 with 8N Ca (OH) ₂・ Membrane permeate flow control: Flow rate control by transmembrane differential pressure (transmembrane differential pressure: controlled at 0.1 kPa to less than 20 kPa)
-The culture tank containing the separation membrane element is autoclaved at 121 ° C for 20 min and autoclaved.

  The HPLC shown in Reference Example 2 was used for the evaluation of the concentration of lactic acid as a product, and “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used for the glucose concentration measurement.

  First, HI003 strain was shaken overnight in 5 ml of SC4 medium in a test tube to obtain a culture solution (pre-culture). The obtained culture solution was inoculated into 100 ml of fresh SC4 medium, and cultured with shaking at a temperature of 30 ° C. for 24 hours in a 500 ml Sakaguchi flask (pre-culture). The culture solution is inoculated in a 1.5 L SC4 medium of the membrane separation type continuous fermentation apparatus shown in FIG. 2, and the fermentation reaction tank 1 is stirred at 400 rpm by the attached stirrer 5. Aeration was adjusted, temperature was adjusted, pH was adjusted, and culture was performed for 24 hours (pre-culture). Immediately after completion of the pre-culture, the SC4 medium is continuously supplied, and the continuous culture is performed while controlling the amount of permeated water so that the amount of the fermentation broth of the membrane separation type continuous fermentation apparatus becomes 1.5 L. Manufactured. Control of the amount of permeated water through the continuous fermentation test was performed by appropriately changing the transmembrane differential pressure to be 0.1 kPa or more and less than 20 kPa by the differential pressure control device 3. The produced lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. In addition, the yield of lactic acid versus sugar and the lactic acid production rate calculated from the input glucose calculated from the lactic acid and glucose concentrations were measured.

  Table 6 shows the results of an 800-hour fermentation test. According to the method for producing lactic acid of the present invention using the continuous fermentation apparatus shown in FIG. 2, stable production of lactic acid by continuous fermentation was possible.

(Example 3) Production of lactic acid by continuous fermentation using a high-order polyploid (nutrient-unnecessary) yeast having the ability to produce lactic acid (Part 3)
It implemented and evaluated on the same conditions as Example 1 except having used the porous membrane created in said Reference Example 4 for the separation membrane. Table 6 shows the results of a 750 hour continuous fermentation test. According to the method for producing lactic acid of the present invention, stable production of lactic acid by continuous fermentation was possible.

(Example 4) Production of lactic acid by continuous fermentation using a high-order polyploid (nutrient non-requirement) yeast having the ability to produce lactic acid (Part 4)
The evaluation was performed under the same conditions as in Example 2 except that the porous separation membrane prepared in Reference Example 4 was used as the separation membrane. Table 6 shows the results of a 770 hour fermentation test. Stable production of lactic acid by continuous fermentation was possible by the lactic acid production method of the invention.

(Comparative Example 1) Production of lactic acid by batch fermentation using yeast having the ability to produce lactic acid (nutrient-neutral requirement) Yeast is the most typical batch fermentation as a fermentation form using bacterial cells, L-lactic acid productivity was evaluated. Using the SC4 medium shown in Table 3, a batch fermentation test was conducted using only the fermentation reaction tank 1 of the membrane separation type continuous fermentation apparatus of FIG. The medium was used after autoclaving at 121 ° C. for 15 minutes. Also in Comparative Example 1, the yeast HI003 strain prepared in Reference Example 1 described above was used as the bacterial cell, and the HPLC shown in Reference Example 2 above was used to determine the concentration of the product lactic acid and the saccharides in the culture solution. Was used.

The operating conditions of Comparative Example 1 are shown below.
・ Fermentation reactor capacity: 2 (L)
・ Temperature adjustment: 30 (℃)
-Aeration volume of fermentation reaction tank: 0.05 (L / min)
・ Fermentation reactor stirring speed: 100 (rpm)
・ PH adjustment: pH adjusted to 5 with 8N Ca (OH) ₂・ Culture tank is autoclaved at 121 ° C. for 20 min and autoclaved with high pressure steam.

  First, the HI003 strain was cultured with shaking in a 5 ml lactic acid fermentation medium in a test tube overnight (pre-culture). The culture solution was inoculated into 100 ml of fresh lactic acid fermentation medium and cultured with shaking in a 500 ml Sakaguchi flask for 24 hours (pre-culture). The preculture liquid was inoculated into 1 L of a lactic acid fermentation medium of a membrane separation type continuous fermentation apparatus, the fermentation reaction tank 1 was stirred at 100 rpm by the attached stirrer 5, and the fermentation reaction tank 1 was aerated. Temperature adjustment and pH adjustment were performed, and batch fermentation culture was performed without operating the fermentation culture medium circulation pump 11. The amount of bacterial cell growth at this time was 14 in terms of absorbance at 600 nm. The results of batch fermentation are shown in Table 6. Fermentation time was short, and lactic acid to sugar yield and lactic acid production rate were inferior to the examples of the present invention.

(Comparative Example 2) Production of lactic acid by continuous fermentation using haploid yeast (nutrient non-requirement) (Part 1)
Evaluation was performed under the same conditions as in Example 1 except that the yeast HI003-1B strain prepared in Reference Example 1 was used. As a result, the transmembrane pressure difference became 20 kPa or more in 600 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 6. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 3) Production of lactic acid by continuous fermentation using haploid yeast (nutrient non-requirement) (Part 2)
The evaluation was performed under the same conditions as in Example 2 except that the yeast HI003-1B strain prepared in Reference Example 1 was used. As a result, the transmembrane pressure difference became 20 kPa or more in 600 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 6. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 4) Production of lactic acid by continuous fermentation using haploid yeast (nutrient non-requirement) (Part 3)
The evaluation was performed under the same conditions as in Example 3 except that the yeast HI003-1B strain prepared in Reference Example 1 was used. As a result, the transmembrane pressure difference became 20 kPa or more in 500 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 6. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 5) Production of lactic acid by continuous fermentation using haploid yeast (no nutrient requirement) (Part 4)
The evaluation was carried out under the same conditions as in Example 4 except that the yeast HI003-1B strain prepared in Reference Example 1 was used. As a result, the transmembrane pressure difference became 20 kPa or more in 500 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 6. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 6) Production of lactic acid by continuous fermentation using haploid yeast (nutrient non-requirement) (Part 1)
It implemented and evaluated on the same conditions as Example 1 except having used the yeast SU014-3B strain | stump | stock produced by the reference example 1. FIG. As a result, the transmembrane pressure difference became 20 kPa or more in 300 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 6. As a result, it has been clarified that lactic acid can be produced stably over a long period of time by using non-nutrient-requiring yeast or higher polyploid yeast.

(Comparative Example 7) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) (Part 2)
It implemented and evaluated on the same conditions as Example 2 except having used the yeast SU014-3B strain | stump | stock produced by the reference example 1. FIG. As a result, the transmembrane pressure difference became 20 kPa or more in 280 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 6. As a result, it has been clarified that lactic acid can be produced stably over a long period of time by using non-nutrient-requiring yeast or higher polyploid yeast.

(Comparative Example 8) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) (Part 3)
The test was carried out and evaluated under the same conditions as in Example 3 except that the yeast strain SU014-3B prepared in Reference Example 1 was used. As a result, the transmembrane pressure difference became 20 kPa or more in 350 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 6. As a result, it has been clarified that lactic acid can be produced stably over a long period of time by using non-nutrient-requiring yeast or higher polyploid yeast.

(Comparative Example 9) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) (Part 4)
It implemented and evaluated on the same conditions as Example 4 except having used the yeast SU014-3B strain | stump | stock produced by the reference example 1. FIG. As a result, the transmembrane pressure difference became 20 kPa or more in 270 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 6. As a result, nutritional unsolicited yeast also properly was found to be able to produce lactic acid preferable to use a high following polyploid yeast stably for a long period of time.

(Example 5) Production of lactic acid by continuous fermentation using a high-order polyploid (nutrient non-requirement) yeast having the ability to produce lactic acid (part 5)
The evaluation was performed under the same conditions as in Example 1 except that the lactic acid fermentation medium shown in Table 4 was used. Table 7 shows the results of an 800-hour fermentation test. A lactic acid fermentation medium having fewer nutrient sources than the SC4 medium could also be produced by continuous fermentation of stable lactic acid by the lactic acid production method of the present invention.

(Example 6) Production of lactic acid by continuous fermentation using a higher polyploidy (nutrient non-requirement) yeast having the ability to produce lactic acid (Part 6)
The evaluation was carried out under the same conditions as in Example 2 except that the lactic acid fermentation medium shown in Table 4 was used. Table 7 shows the results of an 800-hour fermentation test. A lactic acid fermentation medium having fewer nutrient sources than the SC4 medium could also be produced by continuous fermentation of stable lactic acid by the lactic acid production method of the present invention.

(Example 7) Production of lactic acid by continuous fermentation using a high-order polyploid (nutrient-unnecessary) yeast having the ability to produce lactic acid (Part 7)
The evaluation was performed under the same conditions as in Example 3 except that the lactic acid fermentation medium shown in Table 4 was used. Table 7 shows the results of an 800-hour fermentation test. A lactic acid fermentation medium having fewer nutrient sources than the SC4 medium could also be produced by continuous fermentation of stable lactic acid by the lactic acid production method of the present invention.

(Example 8) Production of lactic acid by continuous fermentation using high-order polyploid (nutrient non-requirement) yeast having the ability to produce lactic acid (No. 8)
The evaluation was performed under the same conditions as in Example 4 except that the lactic acid fermentation medium shown in Table 4 was used. Table 7 shows the results of an 800-hour fermentation test. A lactic acid fermentation medium having fewer nutrient sources than the SC4 medium could also be produced by continuous fermentation of stable lactic acid by the lactic acid production method of the present invention.

(Example 9) Production of lactic acid by continuous fermentation using high-order polyploid yeast (nutritional requirement) having the ability to produce lactic acid (Part 1)
The test was carried out and evaluated under the same conditions as in Example 5 except that the yeast SU014 strain prepared in Reference Example 1 was used. Table 7 shows the results of a 700-hour continuous fermentation test. Although it was slightly inferior in lactic acid to sugar yield and lactic acid production rate compared to the non-nutrient demanding high-order polyploid yeast, it was possible to produce lactic acid stably for a long period by continuous fermentation.

(Example 10) Production of lactic acid by continuous fermentation using high-order polyploid yeast (nutritional requirement) having the ability to produce lactic acid (Part 2)
The test was carried out and evaluated under the same conditions as in Example 6 except that the yeast SU014 strain prepared in Reference Example 1 was used. The results of a 700 hour fermentation test are shown in Table 7. Although it was slightly inferior in lactic acid to sugar yield and lactic acid production rate compared to the non-nutrient demanding high-order polyploid yeast, it was possible to produce lactic acid stably for a long period by continuous fermentation.

(Example 11) Production of L-lactic acid by continuous fermentation using high-order polyploid yeast (nutrient requirement) having the ability to produce lactic acid (part 3)
The test was carried out and evaluated under the same conditions as in Example 7 except that the yeast SU014 strain prepared in Reference Example 1 was used. Table 7 shows the results of a 650 hour continuous fermentation test. Although it was slightly inferior in lactic acid to sugar yield and lactic acid production rate compared to the non-nutrient demanding high-order polyploid yeast, it was possible to produce lactic acid stably for a long period by continuous fermentation.

(Example 12) Production of lactic acid by continuous fermentation using high-order polyploid yeast (nutrient requirement) having the ability to produce lactic acid (Part 4)
The test was carried out and evaluated under the same conditions as in Example 8 except that the yeast SU014 strain prepared in Reference Example 1 was used. Table 7 shows the results of a 670 hour fermentation test. Although it was slightly inferior in lactic acid to sugar yield and lactic acid production rate compared to the non-nutrient demanding high-order polyploid yeast, it was possible to produce lactic acid stably for a long period by continuous fermentation.

(Comparative Example 10) Production of lactic acid by batch fermentation using lactic acid-producing higher-order polyploid (nutrient-unnecessary) yeast Same as Comparative Example 1 except that the lactic acid fermentation medium shown in Table 4 was used. Conducted and evaluated under conditions. The results of batch fermentation are shown in Table 7. Fermentation time was short, and lactic acid to sugar yield and lactic acid production rate were inferior to the examples of the present invention.

(Comparative Example 11) Production of lactic acid by batch fermentation using a high-order polyploid yeast (nutritional requirement) having the ability to produce lactic acid Same as Comparative Example 2 except that the SU014 strain prepared in Reference Example 1 was used. Conducted and evaluated under conditions. The results of batch fermentation are shown in Table 7. Fermentation time was short, and lactic acid to sugar yield and lactic acid production rate were inferior to the examples of the present invention.

(Comparative Example 12) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) having the ability to produce lactic acid (Part 1)
It implemented and evaluated on the same conditions as Example 5 except having used yeast SU014-3B strain | stump | stock produced by the reference example 1. FIG. As a result, the transmembrane pressure difference became 20 kPa or more in 300 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 7. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 13) Production of lactic acid by continuous fermentation using haploid yeast (nutrient requirement) having the ability to produce lactic acid (Part 2)
It implemented and evaluated similarly to Example 6 except having used the yeast SU014-4B strain | stump | stock produced by the reference example 1. FIG. As a result, the transmembrane pressure difference became 20 kPa or more in 280 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 7. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 14) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) having the ability to produce lactic acid (Part 3)
It implemented and evaluated similarly to Example 7 except having used yeast SU014-3B strain | stump | stock produced by the reference example 1. FIG. As a result, in the case of haploid yeast, the transmembrane pressure difference became 20 kPa or more in 350 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 7. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 15) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) having the ability to produce lactic acid (Part 4)
It implemented and evaluated similarly to Example 8 except having used the yeast SU014-3B strain | stump | stock produced by the reference example 1. FIG. As a result, the transmembrane pressure difference became 20 kPa or more in 270 hours, and the culture was terminated. The continuous fermentation test results are shown in Table 7. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Example 13) Production of lactic acid by continuous fermentation using high-order polyploidy (nutrient non-requirement) yeast having the ability to produce lactic acid (No. 9)
Membrane separation element effective filtration area was expanded to 240 cm ² , and the same conditions as in Example 1 were used and evaluated except that the lactic acid fermentation medium 2 shown in Table 5 was used. The results of a 1400 hour fermentation test are shown in FIGS. According to the method for producing lactic acid of the present invention, it was possible to produce lactic acid stably for a long time by continuous fermentation.

(Example 14) Production of lactic acid by continuous fermentation using a higher-order polyploid (nutrient-unnecessary) yeast having the ability to produce lactic acid (No. 10)
Membrane separation element effective filtration area was expanded to 240 cm ² , and the same conditions as in Example 3 were used and evaluated except that the lactic acid fermentation medium 2 shown in Table 5 was used. The results of a 1350 hour fermentation test are shown in FIGS. According to the method for producing lactic acid of the present invention, it was possible to produce lactic acid stably for a long time by continuous fermentation.

(Example 15) Production of lactic acid by continuous fermentation using a higher-order polyploid (nutrient-unnecessary) yeast having the ability to produce lactic acid (Part 11)
The tests were performed and evaluated under the same conditions as in Example 13 except that the yeast SE001 strain prepared in Reference Example 1 was used. The results of a 1350 hour fermentation test are shown in FIGS. According to the method for producing lactic acid of the present invention, it was possible to produce lactic acid stably for a long time by continuous fermentation.

(Example 16) Production of lactic acid by continuous fermentation using a high-order polyploid (nutrient-unnecessary) yeast having the ability to produce lactic acid (No. 12)
The test was carried out and evaluated under the same conditions as in Example 14 except that the yeast SE001 strain prepared in Reference Example 1 was used. The results of a 1350 hour fermentation test are shown in FIGS. According to the method for producing lactic acid of the present invention, it was possible to produce lactic acid stably for a long time by continuous fermentation.

(Comparative Example 16) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) having the ability to produce lactic acid (Part 5)
The test was carried out and evaluated under the same conditions as in Example 13 except that the yeast strain SU014-3B prepared in Reference Example 1 was used. As a result, the transmembrane pressure difference became 20 kPa or more in 300 hours, and the culture was terminated. The continuous fermentation test results are shown in FIGS. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 17) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) having the ability to produce lactic acid (Part 6)
The test was carried out and evaluated under the same conditions as in Example 14 except that the yeast strain SU014-3B prepared in Reference Example 1 was used. As a result, the transmembrane pressure difference became 20 kPa or more in 280 hours, and the culture was terminated. The continuous fermentation test results are shown in FIGS. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 18) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) having the ability to produce lactic acid (Part 7)
It implemented and evaluated on the same conditions as Example 15 except having used yeast SE001-1A strain | stump | stock produced by the reference example 1. FIG. As a result, the transmembrane pressure difference became 20 kPa or more in 300 hours, and the culture was terminated. The continuous fermentation test results are shown in FIGS. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

(Comparative Example 19) Production of lactic acid by continuous fermentation using haploid yeast (nutritional requirement) having the ability to produce lactic acid (No. 8)
It implemented and evaluated on the same conditions as Example 16 except having used yeast SE001-1A strain | stump | stock produced by the reference example 1. FIG. As a result, the transmembrane pressure difference became 20 kPa or more in 270 hours, and the culture was terminated. The continuous fermentation test results are shown in FIGS. As a result, it was revealed that lactic acid can be produced stably over a long period of time by using higher-order polyploid yeast.

  In addition, Table 8 shows the culture conditions of Examples 1 to 16 and Comparative Examples 1 to 19.

  According to the method for producing lactic acid by the continuous fermentation method using the higher order polyploid yeast of the present invention as a test bacterium, high productivity of lactic acid which is a desired fermentation product stably over a long period of time under simple operation conditions. Thus, it is possible to stably produce lactic acid, which is a fermentation product, at a low cost in the fermentation industry.

Claims (9)

A high-order polyploidy yeast -free culture medium having the ability to produce lactic acid into which a lactate dehydrogenase gene has been introduced is filtered through a porous membrane having an average pore size of 0.01 μm or more and less than 1 μm. A method for producing lactic acid by continuous fermentation, wherein the product is recovered from the filtrate, the unfiltered solution is retained or refluxed in the culture solution, and a fermentation raw material is added to the culture solution.   The method for producing lactic acid according to claim 1, wherein the filtration is performed with the intermembrane pressure difference of the porous membrane in the range of 0.1 kPa to less than 20 kPa.   The method for producing lactic acid according to claim 1 or 2, wherein the high-order polyploid yeast is diploid. The method for producing lactic acid according to any one of claims 1 to 3 , wherein the yield of lactic acid to sugar in continuous fermentation is 70% or more. The method for producing lactic acid according to any one of claims 1 to 4 , wherein the lactic acid accumulation concentration in the culture solution of continuous fermentation is 40 g / L or more. The method for producing lactic acid according to any one of claims 1 to 5 , wherein the lactic acid production rate of continuous fermentation is 7.5 g / L / h or more. The method for producing lactic acid according to any one of claims 4 to 6 , wherein continuous fermentation is continued for 400 hours or more. Yeast, characterized in that the belonging to the genus Saccharomyces (Saccharomyces), The method for producing lactic acid according to any one of claims 1-7. The method for producing lactic acid according to any one of claims 1 to 8, characterized in that yeast is Saccharomyces Serebise (Saccharomyces cerevisiae).

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